an immunofluorescence-based method for visualizing tuft cells in jejunum cryosections

An Immunofluorescence-Based Method for Visualizing Tuft Cells in Jejunum Cryosections

To prepare sections of jejunum, start by setting both the cryostat chamber temperature and object temperature to minus 22 degrees Celsius. Place the frozen tissue block in a cryo-mold in the cryostat chamber for at least 15 minutes. After 15 minutes, cut the block in half with a razor blade to expose the transverse section of the jejunum. Mount one half of the block on a cryostat adapter. Section the gelatin-filled jejunum into 30-micron thick sections. Use frozen forceps to gently transfer the sections into a 35-millimeter culture dish containing 3 milliliters of PBS.
After washing the sections three times for 5 minutes each with 3 milliliters of PBS-T, add 3 milliliters of freshly prepared antigen retrieval solution to the dish containing the free-floating sections. Then, close the lid, seal the gap between the dish and the lid with a strip of vinyl tape, and incubate at 50 degrees Celsius in a hybridization incubator for three hours without shaking.
After immunostaining, transfer the dish of stained sections in PBS to a stereoscopic microscope. Place 200 microliters of PBS in a droplet on the center of an AMS-coated white glass slide. Then, use a P200 pipette tip to transfer one jejunum section from the dish into the droplet.
After adjusting the section alignment, aspirate all remaining PBS surrounding the section. Add 20 microliters of aqueous mounting media, and place a coverslip atop the media. Immediately seal the coverslip edges with xylene-based mounting media, and allow to dry for two to three hours before beginning confocal microscopy.

an-immunofluorescence-based-method-for-visualizing-tuft-cells-jejunum

Universidad de Cartagena UDEC

Research

JoVE Encyclopedia of Experiments

Immunology

Antibody-Based Technologies

Antibody-Based Imaging and Detection Methods

EoE Sub Articles

eoe sub articles

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Preparations
<ol>
	<li>Prepare 10 L of phosphate-buffered saline (PBS): 32.27 g of sodium hydrogen phosphate (Na2HPO4&#183;12H2O), 4.5 g of Sodium dihydrogen phosphate (NaH2PO4&#183;2H2O), 80.0 g of sodium chloride (NaCl) added to ultrapure water to a final volume of 10 L. Store solution at room temperature (RT).</li>
	<li>Prepare 200 mL of PBS-T: 200 mL of PBS from step 1.1 with 100 &#181;L of polyoxyethylene (10) octylphenyl ether to a final concentration of 0.05% (vol: vol). Store at RT.</li>
	<li>While wearing gloves and eye protection, prepare 50 mL of 15% sucrose in 10% buffered neutral formalin solution: 7.5 g of sucrose added to 10% buffered neutral formalin solution (Table of Materials) to a final volume of 50 mL. Store at RT. 
	CAUTION: Inhalation and/or skin/eye contact with formaldehyde in 10% buffered neutral formalin solution can be hazardous. Handle with caution.</li>
	<li>Prepare 1.5 mL of 200 units/mL phalloidin-fluorescent dye conjugate stock solution: phalloidin-fluorescent dye conjugate (300 units in 1 vial, lyophilized solids, excitation, and emission wavelengths: 581 nm and 609 nm, respectively) suspended in 1.5 mL of methanol. Store the solution in the dark at -20 &#176;C.</li>
	<li>Prepare 5 mg/mL 4&#39;,6-diamidino-2-phenylindole, dihydrochloride (DAPI) stock solution: 10 mg of DAPI in 2 mL of N, N-dimethylformamide (DMF). Store the solution in the dark at -20 &#176;C.</li>
	<li>Prepare 5% bovine serum albumin (BSA) in PBS: 0.5 g of BSA, 10 mL of PBS. Store at 4 &#176;C.</li>
	<li>Remove the beveled tip of an 18-gauge straight needle using a nipper, and pinch the nipped end with a pincher in a lengthwise direction to allow liquid to flow through the needle lumen (Figure 1A1). 
	NOTE: The protocol can be paused here.</li>
</ol>
2. Animal Dissection and Isolation of Jejunum
<ol>
	<li>Perfusion fixation of a mouse
	<ol>
		<li>Wear gloves and work in a well-ventilated area.</li>
		<li>Prepare a 100 mL glass beaker, 10% buffered neutral formalin solution, surgical tools (scissors, forceps), a metallic tray, 6-0 nylon cut sutures, an 18-gauge straight needle prepared as in 1.7, a 22-gauge winged needle, a 20-mL syringe.</li>
		<li>Load 20 mL of 10% buffered neutral formalin solution from the glass beaker into the 20-mL syringe and attach the 22-gauge winged needle to the outlet.</li>
		<li>Prepare liquid gelatin by adding 1 g of gelatin powder to 20 mL of PBS (final 5% gelatin) in a 50-mL centrifuge tube. After soaking at RT for 15 min, incubate at 50 &#176;C in a water bath for 15 min without shaking.</li>
		<li>Briefly vortex and incubate at 50 &#176;C for another 15 min to completely dissolve the gelatin. Leave the tube at RT until use.</li>
		<li>Euthanize an adult mouse by cervical dislocation.</li>
		<li>Place the mouse from step 2.1.6 on a metallic tray and make a small incision on the skin through the ensiform cartilage using surgical tools.</li>
		<li>Expand the incision with both hands to expose the thoracic and abdominal regions.</li>
		<li>Open the peritoneum to expose the diaphragm, and then open the diaphragm on both sides.</li>
		<li>Cut the thoracic cage along the anterior axillary lines on both sides, then remove the thoracic wall by cutting in a transverse direction at the position of the thymus to expose the heart.</li>
		<li>Make a small incision on the auricle of the right atrium to drain the blood and insert a 22-gauge winged needle into the apex of the left ventricle. Push the plunger to perfuse the tissues with a 10% buffered neutral formalin solution.</li>
		<li>After exposing organs in the pelvis, cut off the end of the rectum from the anus, and separate the intestines from the body by cutting the mesentery.</li>
		<li>To isolate the jejunum, cut the intestines at 4 cm from the anal side of the gastric antrum. Discard the anal half of the remaining small intestine.</li>
		<li>Clip the jejunum in half to facilitate the flushing procedure. Load 20 mL of 10% buffered neutral formalin solution into the 20-mL syringe, and attach the 18-gauge straight needle prepared in step 1.7 to the outlet.</li>
		<li>Inject 10% buffered neutral formalin solution from one end of the clipped jejunum to flush out the intestinal contents and to fix the gut lumen surface (Figure 1A2).</li>
		<li>Wash the gut lumen by injecting PBS as described in step 2.1.15.</li>
		<li>Flush liquid gelatin into the gut lumen as described in step 2.1.15 to replace PBS with liquid gelatin.</li>
		<li>Close one end of the clipped jejunum by suture ligation using a 6-0 nylon cut suture, fill the jejunum with liquid gelatin, and close the opposite end by suture ligation (Figure 1A3).</li>
		<li>Add four suture knots to fit a sausage-shaped jejunum section to the depressed portion of a cryomold (Figure 1A4).&#160;&#160;&#160;&#160; 
		NOTE: For cryomolds with a 20 mm x 25 mm x 5 mm depressed section, a ~20 mm sausage-like jejunum section is preferable.</li>
		<li>Soak the tissues in 50 mL of 15% sucrose in 10% buffered neutral formalin solution overnight at 4 &#176;C.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
		NOTE: These steps ensure cryoprotection by sucrose and protein fixation by formalin of gelatin and tissue. Formalin-gelatinized gelatin does not melt at RT, whereas non-fixed gelatinized gelatin at 4 &#176;C melts at RT.</li>
	</ol>
	</li>
</ol>
3. Snap Freezing of Gelatin-filled Jejunum Tissues
<ol>
	<li>By cutting the jejunum at the suture knots, three sausage-like jejunum pieces with both ends ligated are obtained (Figure 1A4). Align the sausage-like pieces in a cryomold for the addition of embedding compound for the preparation of frozen tissue specimens.</li>
	<li>Snap freeze the cryomold in isopentane cooled with liquid nitrogen. Store cryomolds at -80 &#176;C&#160; 
	NOTE: The protocol can be paused here.</li>
</ol>
4. Immunofluorescence of Tuft Cells Using Free-floating Cryosections
<ol>
	<li>Cryosectioning
	<ol>
		<li>Set both the cryostat chamber temperature (CT) and object temperature (OT) to -22 &#176;C. Place the frozen tissue block in a cryomold in the cryostat chamber for at least 15 min.</li>
		<li>Add 3 mL of PBS to a 35-mm culture dish.</li>
		<li>Remove the frozen tissue block from the cryomold and cut the block in half with a razor blade to expose the transverse section of the jejunum. Mount one half of the block on a chuck (a cryostat adaptor) to section the cutting plane with the razor blade.</li>
		<li>Section the gelatin-filled jejunum into 30 &#181;m-thick sections. Use frozen forceps to gently transfer the sections into the 35-mm culture dish prepared in step 4.1.2 (Figure 2B, right).</li>
	</ol>
	</li>
	<li>Application of primary antibodies
	<ol>
		<li>Wash the free-floating sections in the 35-mm culture dish 3 times for 5 min each with 3 mL PBS-T with mild shaking on a reciprocal shaker (approximately 36 times/min).</li>
		<li>Prepare antigen retrieval solution: 0.3 mL of antigen retrieval solution concentrate (Table of Materials) in 2.7 mL of ultrapure water. Add 3 mL of antigen retrieval solution to the 35-mm culture dish containing free-floating sections.</li>
		<li>Close the lid, seal the gap between the dish and the lid with a strip of vinyl tape, and incubate at 50 &#176;C in a hybridization incubator for 3 h without shaking.</li>
		<li>Remove the dish from the incubator and cool at RT for 20 min. After removing the vinyl tape, wash the sections 3 times for 5 min each with 3 mL of PBS-T and mild shaking.</li>
		<li>Aspirate the PBS-T and add 5 drops of blocking solution (Table of Materials) onto the sections. Incubate at RT for 5 min with mild shaking.</li>
		<li>Prepare the primary antibody solution: 495 &#181;L of 5% BSA in PBS with 5 &#181;L of phospho-Y1798 girdin (pY1798) antibodies (Table of Materials). Add 500 &#181;L of primary antibody solution onto the sections (the blocking solution does not need to be removed).</li>
		<li>Place the dish in a humidified incubation chamber and incubate overnight at 4 &#176;C with mild shaking.&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
		NOTE: The overnight incubation can be extended to up to three nights.</li>
	</ol>
	</li>
	<li>Application of secondary antibodies
	<ol>
		<li>Wash the sections 3 times for 5 min each in 3 mL of PBS-T with mild shaking.</li>
		<li>Prepare diluted DAPI stock solution: 2 &#181;L of DAPI stock solution (Step 1.5), 998 &#181;L of PBS.</li>
		<li>Make secondary antibody solution: 476 &#181;L of 5% BSA in PBS, 10.5 &#181;L of diluted DAPI solution, 12.5 &#181;L of phalloidin-fluorescent dye conjugate stock solution, 1 &#181;L of goat anti-rabbit IgG-fluorescent dye conjugate (wavelength: excitation 496 nm, emission 520 nm).</li>
		<li>After aspirating the PBS-T, apply the secondary antibody solution and incubate in a light-shielded incubation chamber at RT for 30 min with mild shaking.</li>
		<li>Wash the sections 3 times for 5 min each with 3 mL of PBS-T and mild shaking.</li>
		<li>After the final wash, remove the PBS-T and replace it with 3 mL of PBS lacking polyoxyethylene(10) octylphenyl ether. Transfer the dish to a stereoscopic microscope.</li>
		<li>Place 200 &#181;L of PBS in a droplet on the center of a MAS-coated white glass slide and transfer one jejunum section from the dish into the droplet using a P200 pipet tip.</li>
		<li>After adjusting the section alignment under the stereoscopic microscope, aspirate all remaining PBS surrounding the section.</li>
		<li>Add 20 &#181;L of aqueous mounting media and place a 20 x 20 mm coverslip atop the media.</li>
		<li>Immediately seal the coverslip edges with xylene-based mounting media. Place the slide on a wooden mappe and allow the xylene-based mounting media to solidify at RT for 2 - 3 h.&#160;&#160;&#160;&#160;&#160; 
		NOTE: The protocol can be paused here. After the xylene-based mounting media solidifies, the slides can be stored for 2 - 3 weeks in a light-shielded slide box at RT.</li>
	</ol>
	</li>
</ol>
5. Confocal Microscopy
<ol>
	<li>Put immersion oil on the 63X objective of a confocal microscope. Turn the coverslip slide down and place the slide on the stage.</li>
	<li>Digitize the TC images acquired at laser wavelengths 405, 488, and 555 nm and save the images in tiff format (.tiff).&#160;&#160;&#160;&#160;&#160; 
	NOTE: Choose a detection filter set that is appropriate for the fluorescence dyes (i.e., excitation/emission maxima: 358/461 nm, 490/525, and 590/617).</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Slc:DDY 6-week-old female mice</td>
			<td>Chubu Kagaku Shizai</td>
			<td>Not applicable</td>
			<td></td>
		</tr>
		<tr>
			<td>Disodium hydrogenphosphate 12-water (Na2HPO412H2O)</td>
			<td>Wako</td>
			<td>196-02835</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dihydroenphosphate Dihydrate (NaH2PO42H2O</td>
			<td>Wako</td>
			<td>199-02825</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Wako</td>
			<td>199-10665</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100,&#160; Polyoxyethylene(10) octylphenyl ether</td>
			<td>Katayama Chemical</td>
			<td>Not applicable</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Wako</td>
			<td>190-00013</td>
			<td></td>
		</tr>
		<tr>
			<td>10% buffered neutral formalin solution</td>
			<td>Muto Chemical</td>
			<td>20215</td>
			<td>Step 1.3.</td>
		</tr>
		<tr>
			<td>Phalloidin-fluorescent dye conjugate, Alexa Fluor 594 phalloidin</td>
			<td>ThermoFisher Scientific</td>
			<td>A12381</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Nacalai Tesque</td>
			<td>21915-35</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI (4&#39;,6-diamidino-2-phenylindole, dihydrochloride)</td>
			<td>ThermoFisher Scientific</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>N.N-dimethylformamide (DMF)</td>
			<td>Nacalai Tesque</td>
			<td>13015-75</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma</td>
			<td>A9647-10G</td>
			<td></td>
		</tr>
		<tr>
			<td>18-gauge stainless steel straight needle</td>
			<td>Terumo</td>
			<td>NN-1838R</td>
			<td></td>
		</tr>
		<tr>
			<td>U.S.P. 6-0, 60 cm nylon cut suture, Crownjun</td>
			<td>Kono Seisakusho</td>
			<td>Not applicable</td>
			<td></td>
		</tr>
		<tr>
			<td>22-gauge winged needle</td>
			<td>Terumo</td>
			<td>SV-22DLK</td>
			<td></td>
		</tr>
		<tr>
			<td>20 mL TERUMO syringe</td>
			<td>Terumo</td>
			<td>SS-20ES</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL centrifuge tube</td>
			<td>TPP</td>
			<td>91050</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL Iwaki centrifuge tubes</td>
			<td>Iwaki</td>
			<td>2325-015</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL micro test tube</td>
			<td>Star</td>
			<td>RSV-MTT1.5</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin powder, Gelare-blanc</td>
			<td>Nitta Biolab</td>
			<td>2809</td>
			<td></td>
		</tr>
		<tr>
			<td>Embedding compound for frozen tissue specimen, O.C.T. Compound 118 mL 12 piece</td>
			<td>Sakura FineTech</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryomold, CRYO DISH No. 3, 125 piece, well size 20 x 25 x 5 (mm)</td>
			<td>Shoei Work&#39;s</td>
			<td>1101-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopentane</td>
			<td>Nacalai Tesque</td>
			<td>26404-75</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon cell culture dish 35 x 10mm Easy-Grip</td>
			<td>Corning</td>
			<td>353001</td>
			<td></td>
		</tr>
		<tr>
			<td>Antigen retrieval solution concentrate, Target Retrieval Solution pH 9 10x</td>
			<td>Dako</td>
			<td>S2367</td>
			<td>Antigen retrieval solution concentrate in step 4.2.2.</td>
		</tr>
		<tr>
			<td>Protein Block</td>
			<td>Dako</td>
			<td>X0909</td>
			<td>Blocking solution in 4.2.5.</td>
		</tr>
		<tr>
			<td>Phospho-Y1798 girdin (pY1798) antibodies</td>
			<td>Immuno-Biological Laboratories (IBL) = Company X</td>
			<td>28143</td>
			<td>Phospho-Y1798 girdin antibodies in step 4.2.6.</td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488</td>
			<td>ThermoFisher Scientific</td>
			<td>A11034</td>
			<td></td>
		</tr>
		<tr>
			<td>Aquous mounting media, Prolong Gold Antifade Mountant</td>
			<td>ThermoFisher Scientific</td>
			<td>P36930</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips, Matsunami Micro Cover Glass 22 x 22 mm 100 pcs Thickness No.1</td>
			<td>Matsunami Glass</td>
			<td>C022221</td>
			<td></td>
		</tr>
		<tr>
			<td>Entellan New, xylene-based mounting media for microscopy</td>
			<td>Merck Millipore</td>
			<td>107961</td>
			<td></td>
		</tr>
		<tr>
			<td>MAS-coated slide glass white</td>
			<td>Matsunami Glass</td>
			<td>MI-MAS-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Wooden Mappe KO-type</td>
			<td>Shoei Work&#39;s</td>
			<td>99-40007</td>
			<td></td>
		</tr>
		<tr>
			<td>Immersion Oil 518 F Fluorescence Free 20 ml</td>
			<td>Zeiss</td>
			<td>444960</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettes, Pipetman P (P2, P20, P200, P1000)</td>
			<td>Gilson</td>
			<td>F144801, F123600, F123601, F123602</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrapure water production system</td>
			<td>Advantec</td>
			<td>GS-590</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic glove, Star Nitrile Glove</td>
			<td>Star</td>
			<td>RSU-NGVM</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Leica Microsystems</td>
			<td>CM1950</td>
			<td></td>
		</tr>
		<tr>
			<td>Deep freezer, SANYO Ultra Low</td>
			<td>Sanyo</td>
			<td>MDF-382</td>
			<td></td>
		</tr>
		<tr>
			<td>Showcase refrigerator</td>
			<td>Nihon Freezer</td>
			<td>NC-ME50EC</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath, Thermominder SDminiN (to dissolve gelatin at 50 C)</td>
			<td>Taitec</td>
			<td>0068750-000</td>
			<td></td>
		</tr>
		<tr>
			<td>Hybridization incubator (for antigen retrieval at 50 C)</td>
			<td>Taitec</td>
			<td>HB-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubation chamber for immunostaining</td>
			<td>Cosmo Bio</td>
			<td>10DO</td>
			<td></td>
		</tr>
		<tr>
			<td>Reciprocal shaker for immunohistochemistry (for room temperature)</td>
			<td>Taitec</td>
			<td>NR-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Reciprocal shaker for immunohistochemistry (for 4 C)</td>
			<td>Tokyo Rika Kikai</td>
			<td>MMS-3010</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereoscopic microscope (for tissue handling)</td>
			<td>Olympus</td>
			<td>SZ61</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereoscopic microscope (for Figure 1B)</td>
			<td>Leica Microsystems</td>
			<td>M165FC</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence microscope (for Figure 3)</td>
			<td>Nikon</td>
			<td>E800</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal laser-scanning microscope</td>
			<td>Zeiss</td>
			<td>LSM700</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Mizutani, Y. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/57475">Use of Anti-phospho-girdin Antibodies to Visualize Intestinal Tuft Cells in Free-Floating Mouse Jejunum Cryosections.</a>&#160;J. Vis. Exp. (2018)
This video illustrates an immunofluorescence-based method for visualizing intestinal tuft cells in free-floating mouse jejunum cryosections. Within the intestinal villi, the co-localization of green fluorescence at the luminal tip with an extended red rootlet mass indicates the presence of tuft cells.

<img alt="Figure 1" src="/files/ftp_upload/22102/22102_Figure1.jpg" /> 
Figure 1: Gelatin filling of jejunum sections for morphological preservation of cryosections 
(A)&#160;Photographs of procedures for intraluminal filling of mouse jejunum with gelatin.&#160;(A1)&#160;The beveled tip of an 18-gauge straight needle was removed to avoid piercing the gut wall.&#160;(A2)&#160;Buffered neutral formalin solution (1...

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Begin with a culture dish containing formalin-fixed, gelatin-filled mouse jejunum cryosections featuring tuft cells with apical microvilli and spool-shaped soma.
Wash with a detergent-containing buffer to permeabilize the cell membranes.
Add an alkaline antigen retrieval solution. Incubate to break fixation-induced protein crosslinks, revealing the proteins for staining.
Apply a blocking solution to prevent non-specific antibody binding.
Overlay with primary antibodies targeting an actin-binding protein, specifically phosphorylated in tuft cells.
Remove unbound antibodies. Introduce a mix of fluorophore-conjugated secondary antibodies, DNA-binding dye, and phalloidin-fluorescent dye conjugate.
The secondary antibodies bind to primary antibodies, the phalloidin-fluorescent dye conjugate labels actin, and the DNA-binding dye marks cell nuclei.
Remove unbound components and add a detergent-free buffer.
Transfer the section to an adhesive-coated glass slide, mounting it with media.
Under a confocal microscope, green spool-shaped cells with intensified fluorescence at the luminal tip and an extended red rootlet mass indicate the presence of tuft cells.

28 조회수. Jejunum cryosections에서 Tuft 세포를 시각화하기 위한 면역형광 기반 방법

비디오: Jejunum cryosections에서 Tuft 세포를 시각화하기 위한 면역형광 기반 방법 - 실험

Organ Culture and Whole Mount Immunofluorescence Staining of Mouse Wolffian Ducts

Tubal morphogenesis is a fundamental requirement for the development of most mammalian organs, including the male reproductive system. The epididymis, an integral part of the male reproductive tract, is responsible for sperm storage, maturation, and transport. The adult epididymis is a highly coiled tube that develops from a simple and straight embryonic precursor known as Wolffian duct (WD). Proper coiling of the epididymis is essential for male fertility, as sperm in the testis are unable to fertilize an oocyte. However, the mechanism responsible for epididymal development and coiling remains unclear, partially due to the lack of whole organ culture and imaging methods. In this study, we describe an in vitro culture system and whole mount immunofluorescence protocol to better visualize the process of WD coiling and development, which may also be applied to study other tubular organs.

We present a method for isolation and culture of the mouse Wolffian duct (WD). We also demonstrate a detailed procedure for whole mount immunostaining of cultured/freshly isolated WDs with fluorescently tagged antibodies. Together, these techniques enable the study of WD development, coiling, and differentiation.

오르간 문화와 마우스 Wolffian 덕트의 전체 마운트 면역 형광 염색법

Tubal morphogenesis is a fundamental requirement for the development of most mammalian organs, including the male reproductive system. The epididymis, an ...

연구

JoVE Journal

발생학

Isolation and Quantitative Evaluation of Brush Cells from Mouse Tracheas

Tracheal brush cells are cholinergic chemosensory epithelial cells poised to transmit signals from the airway lumen to the immune and nervous systems. They are part of a family of chemosensory epithelial cells which include tuft cells in the intestinal mucosa, brush cells in the trachea, and solitary chemosensory and microvillous cells in the nasal mucosa. Chemosensory cells in different epithelial compartments share key intracellular markers and a core transcriptional signature, but also display significant transcriptional heterogeneity, likely reflective of the local tissue environment. Isolation of tracheal brush cells from single cell suspensions is required to define the function of these rare epithelial cells in detail, but their isolation is challenging, potentially due to the close interaction between tracheal brush cells and nerve endings or due to airway-specific composition of tight and adherens junctions. Here, we describe a procedure for isolation of brush cells from mouse tracheal epithelium. The method is based on an initial separation of tracheal epithelium from the submucosa, allowing for a subsequent shorter incubation of the epithelial sheet with papain. This procedure offers a rapid and convenient solution for flow cytometric sorting and functional analysis of viable tracheal brush cells.

Brush cells are rare cholinergic chemosensory epithelial cells found in the na&#239;ve mouse trachea. Due to their limited numbers, ex vivo evaluation of their functional role in airway immunity and remodeling is challenging. We describe a method for isolation of tracheal brush cells by flow cytometry.

마우스 기관에서 브러시 셀의 격리 및 정량 평가

Tracheal brush cells are cholinergic chemosensory epithelial cells poised to transmit signals from the airway lumen to the immune and nervous systems. ...

면역학 및 감염병학

Using En Face Immunofluorescence Staining to Observe Vascular Endothelial Cells Directly

Aberrant changes in endothelial phenotype and morphology are considered to be initial events in the pathogenesis of atherosclerosis. Direct observation of the intact endothelium will provide valuable information for understanding the cellular and molecular events in the dysfunctional endothelial cells. Here, we describe a modified en face immunofluorescence staining technique which enables scientists to obtain clear images of the intact endothelial surface and analyze the molecule expression patterns in situ. The method is simple and reliable for observing the entire endothelial monolayer at different sites of the aorta. This technique may be a promising tool for understanding the pathophysiology of atherosclerosis, especially at an early stage.

Here, we present a protocol for immunofluorescence staining to observe the endothelial cells of the mouse aorta directly. This technique is useful when studying the cellular and molecular phenotype of endothelial cells in different flow patterns and in the development of atherosclerosis.

혈관 내피 세포를 직접 관찰하기 위해 En Face 면역 형광 염색사용

Aberrant changes in endothelial phenotype and morphology are considered to be initial events in the pathogenesis of atherosclerosis. Direct observation of ...

An Immunofluorescence-Based Method for Visualizing Tuft Cells in Jejunum Cryosections

내레이션 대본

An Immunofluorescence-Based Method for Visualizing Tuft Cells in Jejunum Cryosections